In vitro screening of cellular events using 3d cell culture systems

ABSTRACT

The present invention relates to in vitro cell culture conditions wherein transfected cells containing promoter-reporter constructs are cultivated in a 3D tissue-like environment. The referred 3D culture conditions may eventually implement appropriate biomaterial or scaffolds.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the priority of provisional patent application60/388,483, filed Jun. 13, 2002, the disclosure of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to in vitro cell culture conditionswherein transfected cells containing one or more selectedpromoter-reporter constructs are cultivated under conditions mimickingthe natural in vivo environment. Such conditions may be achieved byproviding 3D cell arrangements that optionally may include any scaffoldor biomaterial.

The present invention further relates to a non-destructive and real-timeassay for screening various cell types, preferably for cells ofmusculoskeletal tissues or cells being able to differentiate in suchtissues, using key marker genes in form of novel promoter-reporterconstructs that are transfected in said cells.

The present invention further presents small-scale in vitro cell cultureconditions wherein said cell culture conditions have been adapted tovarious multiwell plates in order to enable higher throughputapplications by an easy and convenient read-out with conventionalstandard plate readers or automated confocal microscope reader.

BACKGROUND ART

General Information about Gene-Reporter Technologies

Cellular screening assays are widely used to study eukaryotic geneexpression and cellular physiology such as ELISA-type assays, cellularCa²⁺ assays, cellular assays based on novel fluorescence imaging methodsor expression-reporter gene assays. Especially for reporter-genetechnology the future is estimated to look bright (Naylor et al., 1999).

To a large extent, cellular screening techniques have been developed forpharmaceutical companies to cope with the steadily increasing amount ofcompounds. Various kinds of homogenous high throughput assay systemshave enabled to increase the rate of sample processing (U.S. Pat. No.5,989,835). Unfortunately, focus has mainly been laid on practicabilityrather than on biological relevance. As a consequence, almost allscreening assays use in vitro culture conditions that do not mimicthree-dimensional tissue-like arrangement as it occurs in vivo. Forinstance, disposable plastics have become the preferred substrate usedin modern-day 2D cell cultures. While the growth of cells in twodimensions is a convenient method for preparing, observing and studyingcells in culture, allowing a high rate of cell proliferation, it lacksthe cell-cell and cell-matrix interactions characteristic of wholetissue in vivo. Therefore, these cells will start to loose theirdifferentiated phenotype and become so called de-differentiated cellsand at the same time will start to change their gene expression profile.As a consequence, mimicking tissue-like conditions by e.g. implementingan appropriate biomaterial or scaffold for cellular screening of aspecific cell type is crucial for building up a functional relevant andmeaningful assay. The invention described herein discloses such cellculture conditions that provide an excellent basis for functionalcellular screening by combining appropriate natural or syntheticbiomaterials together with a key set of novel promoter-reporterconstructs.

Background Information Related to Cellular Screening with Cells fromMusculoskeletal Tissues

Focus is being put more and more on cells that can be used to repair orregenerate musculoskeletal tissues such as bone and cartilage by meansof tissue-engineering since they are structurally less complex thanheart valves or entire organs such as pancreas and liver. In case ofpathological conditions they may serve as cellular targets for screeningof more effective drugs. Research efforts, either for tissue-engineeringor for drug screening, in general have to consider differentiationpathways and subsequent maintenance of a differentiated phenotype. Aseries of factors (e.g. growth factors, pharmaceutical agents,biomaterials, mechanical stimulation and others) as well as theirinteractions are critical in this aspect. As a consequence,understanding of the exerted effects and the targeted control by usingthe appropriate factors in the appropriate concentration at the propertime point is far away from being realized. To overcome today's trialand error approaches, it is important to introduce novel and moresophisticated approaches to study this very complex subject matter.

For instance, two recent publications by Grant et al., 2000 and Bergwitzet al., 2001 have been proven to be valuable in tracking cellularresponses of chondrocytes using the rat collagen type II (COL2) promoterin combination with green fluorescent protein (GFP). While Grant et al.generated a COL2-GFP reporter mouse model as a new tool to studyskeletal development by marking the chondrocyte lineage andchondrogenesis; Bergwitz et al. established a stable transfectedchondrocytic cell line and sandwich co-cultures where wnt-secretingcells in monolayer were overlaid by agarose suspension culturescontaining the rat COL2-GFP transfected cell line. In this manner, itwas possible to examine the effect of wnt-proteins on the earlychondrocyte differentiation. Beyond dispute of the scientific impact ofthese publications, broad and simple application of these models is notpossible for several reasons. Only a few laboratories have the tools toproduce transgenic mice, to keep them in animal houses, or analyze themappropriately. Common use is further restricted through a limited numberof experiments, a limited number of promoter-reporter genes and therestriction to mouse species. The chondrogenic rat calvaria cell lineeliminates the above-mentioned restriction with respect to the number ofexperimental testing. However, the drawback of only onepromoter-reporter gene construct and the restriction to a rodent derivedcell line still remains. The narrow scope of application is furtherunderlined by a complex 3D co-culture model requiring quite a high cellnumber which is not suitable for fast and convenient read-outs.

While U.S. Pat. No. 5,932,459 and other literature references, e.g.Stokes et al., 2001, Hauselmann et al., 1994, in principle describeculture systems that could be used to study the re-differentiationprocess of cells they do not allow to be applied within high throughputscreening applications since they all require out of scale quantities ofa characterized cell source. Also the possibility of a read-out processby using conventional plate-readers for such cell-based assays has notbeen addressed.

Furthermore, since all ex vivo tissue-engineering approaches include acell source as one major, if not the most important element, appropriatequality control of current commercialized products is becoming more andmore a central issue. Beyond viability and sterility, it is desired toreceive information about the cellular status of the cells that are useddirectly for a cellular therapy or being used to form de novo tissuefollowing cultivation in 3D.

All these drawbacks have precipitated the concept of monitoring geneexpression of key marker genes within various more functionally relevantculture conditions that allow fast and simple read-out, preferably in anon-destructive and real-time manner.

The current invention offers a new tool to study marker (positive andnegative) gene expression and thus to determine whether the desiredcellular phenotype is maintained.

DISCLOSURE OF THE INVENTION

In a first aspect, the present invention provides a screening method forcompounds having a modulating effect on cellular development and/or celldifferentiation and/or cellular processes. Said screening methodcomprises the following steps:

-   a) cultivating cells harboring a promoter-reporter construct in a 3D    micro-culture under conditions mimicking the natural environment (3D    tissue-like) of said cells, or cultivating said cell in a 2D culture    on bioinductive material,-   b) contacting said cells with a test compound and comparing the    read-out of the promoter-reporter construct to a control.

In a preferred embodiment of the present invention said 3D culturecomprises a biomaterial substrate or scaffold that promotes normalphysiological activity, in particular scaffolds/biomaterials selectedfrom the group of natural scaffolds/biomaterials consisting of alginate,agarose, hyaluronic acid, collagen, proteoglycan, mixtures thereof orfrom the group of synthetic scaffolds/biomaterials consisting ofSkelite™, polyHEMA, polyglycolic acid (PGA), polylactic acid (PLA),mixtures of PGA and PLA.

In a further preferred embodiment said cells are selected from the groupconsisting of chondrocytes, bone cells, rheumatoid cells, osteoarthriticchondrocytes, stem cells, mesenchymal cells, cartilage or bone tumorcells, preferably said cells stem from humans.

In yet a further preferred embodiment said promoter is selected from thegroup consisting of COL1, COL2, SOX9, COMP, MMP2 and aggrecanase-1.

In a further preferred embodiments said reporter is selected from thegroup of GFP, luciferase, β-galactosidase, chloramphenicolacetyltransferase gene (CAT).

Preferred cells for use in a method of the present invention stem fromhumans and said promoter-reporter construct is a DNA construct of thepresent invention.

In a preferred embodiment, said cells comprise more than onepromoter-reporter construct.

Test compounds are preferably selected from the group consisting ofchemical libraries, natural product libraries, peptide libraries, cDNAlibraries and combinatorial libraries.

In a much preferred embodiment of the present invention the method isperformed in a multiplate culture format e.g. 96 or 384-mulitwells.

In a further preferred embodiment said cells are contacted with anactivator or suppressor of said promoter and with a test compound.

In a second aspect, the present invention relates to a DNA construct forcell transfection. Said DNA construct comprises a reporter gene undercontrol of a human promoter wherein said promoter is selected from thegroup consisting of human COL1, human COL2, human SOX9, human COMP,human MMP2 and human aggrecanase-1 and said reporter gene encodes aprotein with an activity that can be detected by colorimetric orfluorescent methods.

In a preferred embodiment said reporter is selected from the groupconsisting of GFP, luciferase, β-galactosidase, chloramphenicolacetyltransferase gene (CAT).

In a further aspect, the present invention relates to a method fortesting whether a material has bioinductive characteristics. Said methodcomprises the following steps:

-   -   culturing cells harboring a promoter-reporter construct on the        material to be tested and    -   comparing the read-out of the promoter-reporter construct to a        control.

In a further aspect, the present invention relates to a method fortesting whether a biomaterial is degraded or resorbed in vivo or invitro. Said method comprising the following steps:

-   -   culturing cells harboring a promoter-reporter construct on the        material to be tested and    -   monitoring expression of the reporter gene in said cells.

In a further aspect, the present invention relates to a use of apromoter-reporter construct for the construction of transgenic animals,preferably transgenic mice. Said construct comprises a reporter which isselected from the group consisting of GFP, luciferase, β-galactosidase,chloramphenicol acetyltransferase gene (CAT) and a promoter which isselected from the group consisting of COL1, COL2, SOX9, COMP, MMP2 andaggrecanase-1.

The resulting transgenic animals can be used in a screening method forcompounds having a modulating effect on cellular development and/or celldifferentiation and/or cellular processes.

In a further aspect the present invention relates to cells or cell linescomprising a reporter construct of the present invention. Said cells orcell lines are preferably selected from the group consisting ofchondrocytes, bone cells, rheumatoid cells, osteoarthritic chondrocytes,stem cells, mesenchymal cells, cartilage or bone tumor cells, preferablysaid cells stem from humans.

In a further aspect, the present invention provides a method for thequality control of cells cultivated in vitro. Said method comprises thefollowing steps:

-   -   transfecting cells that have been cultured in vitro with a key        marker promoter-reporter construct and cultivating said        transfected cells in a 3D culture and    -   detection of the reporter read-out which is indicative for        differentiated cells.

Cells used in said methods are preferably of human origin, preferablycells that belong to the groups as defined herein before. A preferredreporter and preferred promoter for use in said method is selected fromthe groups defined herein before.

The present invention provides a novel cell-based screening assay andvariants thereof that may optionally include biomaterials and scaffolds,either natural or synthetic ones to mimic the environmental nature of agiven tissue within cell culture conditions on a small-scale level. Mostpreferably, the disclosed cell culture conditions in combination withtransfected cells is suitable to cultivate cells derived frommusculoskeletal tissues or cells being able to differentiate in suchtissues.

The present invention also encompasses various novel humantranscriptional promoter-reporter constructs, preferably with thosepromoters that are regarded as key markers for a specific cell type,e.g. collagen type II, SOX9 and COMP for chondrocytes, or a specificcellular status, e.g. aggrecanase-1 (ADAMTS4) and MMP2 forosteoarthritic cells. These gene-reporter constructs, preferably withthe most common luminophore reporters such as GFP or luciferase allowingnon-invasive monitoring of gene expression, can be used in combinationwith the disclosed 3D in vitro cell culture conditions to evaluate theinfluence of signaling molecules, drugs or other medium components onproliferation, differentiation or de novo tissue formation. By usingpluripotent stem cells or progenitor cells, monitoring along adifferentiation pathway or commitment to a specific lineage is possible,most preferably for those cells differentiating into musculoskeletaltissues. Novel functional data about genes having a regulatory rolewithin any of the mentioned cell types can be achieved by e.g.co-transfection of any cell type with gene libraries containing CMVdriven cDNA sequences.

A further important aspect of the invention is the specific adjustmentof the described 3D culture conditions to conventional microtiterplates, e.g. 96 or 384-well format allowing reliable and accurateread-outs with the most important reporter genes such as GFP andluciferase.

A further important aspect of the invention discloses the possibility ofthe described invention to be used as a new quality control tool and/ordiagnostic tool to enhance clinical outcome ofcellular/tissue-engineered therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annexed drawings, wherein:

FIG. 1, time dependent curve for 2×10⁵ adenovirus infected porcinechondrocytes (P2) expressing CMV-GFP in 3D alginate disc culture withina 96-well plate over time, transfection rate 90%. GFP expressionmeasured with BMG Fluostar, 485/20 nm excitation filter and 535/20 nmemission filter during 12 days. ⋄ transfected cells, ▪ control cells.

FIG. 2, cell number dependent curve for adenovirus infected porcinechondrocytes (P2) expressing CMV-GFP in 3D alginate disc culture withina 96-well plate, transfection rate 90%. GFP expression measured with BMGFluostar, 485/20 nm excitation filter and 535/20 nm emission filter atday 2.

FIG. 3, adenovirus infected porcine chondrocytes (P2) expressing CMV-GFPin 3D alginate disc culture within a 96-well plate. a) Image taken bylight microscope on day 3. b) Image taken by fluorescence microscopewith a 470/40 nm excitation filter, a 495 nm beamsplitter and a 525/50nm emission filter on day 3.

FIG. 4, calcein AM/propidium iodide life/dead staining of untransfectedporcine chondrocytes (P2) in 3D alginate disc culture within a 96-wellplate. a) Image taken of living cells by fluorescence microscope with a470/40 nm excitation filter, a 495 nm beamsplitter and a 525/50 nmemission filter on day 12. b) Image taken of dead cells by fluorescencemicroscope with a 535/50 nm excitation filter, a 580 nm beamsplitter anda 590LP nm emission filter on day 12.

FIG. 5, time dependent curve for 2×10⁵ transfected porcine chondrocytes(P2) expressing CMV-GFP in 3D agarose disc culture within 96-well plateover time, transfection rate 90%. GFP expression measured with BMGFluostar, 485/20 nm excitation filter and 535/20 emission filter during13 days. ⋄ transfected cells, ▪ control cells.

FIG. 6, cell number dependent curve for adenovirus infected porcinechondrocytes (P2) expressing CMV-GFP in 3D agarose disc culture within a96-well plate, transfection rate 90%. GFP expression measured with BMGFluostar, 485/20 nm excitation filter and 535/20 nm emission filter atday 2.

FIG. 7, adenovirus transfected porcine chondrocytes (P2) expressingCMV-GFP in 3D agarose disc culture within a 96-well plate. a) Imagetaken by light microscope on day 1. b) Image taken by fluorescencemicroscope with a 470/40 nm excitation filter, a 495 nm beamsplitter anda 525/50 nm emission filter on day 1.

FIG. 8, calcein AM/propidium iodide life/dead staining of untransfectedporcine chondrocytes (P2) in 3D agarose disc culture within a 96-wellplate. a) Image taken of living cells by fluorescence microscope with a470/40 nm excitation filter, a 495 nm beamsplitter and a 525/50 nmemission filter on day 13. b) Image taken of dead cells by fluorescencemicroscope with a 535/50 nm excitation filter, a 580 nm beamsplitter anda 590LP nm emission filter on day 13.

FIG. 9, time dependent curve for 2×10⁵ with Amaxa Nucleofector™technology transfected human chondrocytes (P2) expressing CMV-GFP in 3Dagarose disc culture within a 96-well plate over time, transfection rate40%. GFP expression measured with BMG Fluostar, 485/20 nm excitationfilter and 535/20 nm emission filter during 10 days. ⋄ transfectedcells, ▪ control cells.

FIG. 10, cell number dependent curve for Amaxa Nucleofector™ technologytransfected human chondrocytes (P2) expressing CMV-GFP in 3D agarosedisc culture within a 96-well plate, transfection rate 40%. GFPexpression measured with BMG Fluostar, 485/20 nm excitation filter and535/20 nm emission filter at day 2.

FIG. 11, cell number dependent curve for Fugene6 transfected porcinechondrocytes (P2) expressing COL1-luciferase in 3D agarose disc culturewithin a 96-well plate, transfection rate 15%. Luciferase expressionmeasured with a Berthold Detection System MPL2 luminometer at day 1.

FIG. 12, time dependent curve for 2×10⁵ adenovirus infected porcinechondrocytes (P2) expressing CMV-GFP seeded on polyHEMA within a96-well-plate, transfection rate 90%. GFP expression measured with BMGFluostar, 485/20 nm excitation filter and 535120 nm emission filterduring 10 days. ⋄ transfected cells, ▪ control cells.

FIG. 13, cell number dependent curve for adenovirus infected porcinechondrocytes (P2) expressing CMV-GFP seeded on polyHEMA within a 96-wellplate, transfection rate 90%. GFP expression measured with BMG Fluostar,485/20 nm excitation filter and 535/20 nm emission filter at day 3.

FIG. 14, adenovirus infected porcine chondrocytes (P2) expressingCMV-GFP seeded on polyHEMA in a 96-well plate. a) Image taken by lightmicroscope on day 1. b) Image taken by fluorescence microscope with a470/40 nm excitation filter, a 495 nm beamsplitter and a 525/50 nmemission filter on day 5.

FIG. 15, adenovirus infected osteoarthritic human chondrocytes (P2)expressing CMV-GFP, 16 hours after infection. Image taken byfluorescence microscope with a 470/40 nm excitation filter, a 495 nmbeamsplitter and a 525/50 nm emission filter.

FIG. 16, growth curve of primary human articular chondrocytes (P2)cultured on Osteologic™ discs and standard tissue culture plastic. Cellcounts by trypan blue exclusion method at day 0, 2, 4 and day 7. ⋄ cellson Osteologic™ disc, ▪ cells on standard tissue culture plastic.

FIG. 17, growth of human chondrocytes on standard tissue culture plasticvs. Osteologic™ discs. Passage 3 cells stained with PAS stain at day 7.a) human chondrocytes on standard tissue culture plastic. b) same cellsgrown on Osteologic™ disc.

FIG. 18, Amaxa Nucleofector™ technology transfected human chondrocytes(P0) expressing SOX9-GFP, transfection rate 35%, showing functionalityof the cloned promoter-reporter construct. Image taken by fluorescencemicroscope with a 470/40 nm excitation filter, a 495 nm beamsplitter anda 525/50 nm emission filter.

FIG. 19, Amaxa Nucleofector™ technology transfected human chondrocytes(P0) expressing COL1-GFP, transfection rate 18%, showing functionalityof the cloned promoter-reporter construct. Image taken by fluorescencemicroscope with a 470/40 nm excitation filter, a 495 nm beamsplitter anda 525/50 nm emission filter.

FIG. 20, Amaxa Nucleofector™ technology transfected human chondrocytes(P0) expressing COL2-GFP, transfection rate 10% showing functionality ofthe cloned promoter-reporter construct. Image taken by fluorescencemicroscope with a 470/40 nm excitation filter, a 495 nm beamsplitter anda 525/50 nm emission filter.

FIG. 21, Amaxa Nucleofector™ technology transfected human chondrocytes(P0) expressing COMP-GFP, transfection rate 39%, showing functionalityof the cloned promoter-reporter construct. Image taken by fluorescencemicroscope with a 470/40 nm excitation filter, a 495 nm beamsplitter anda 525/50 nm emission filter.

FIG. 22, Amaxa Nucleofector™ technology transfected human chondrocytes(P0) expressing aggrecanase-1 (ADAMTS4)-GFP, transfection rate 37%,showing functionality of the cloned promoter-reporter construct. Imagetaken by fluorescence microscope with a 470/40 nm excitation filter, a495 nm beamsplitter and a 525/50 nm emission filter.

FIG. 23, Amaxa Nucleofector™ technology transfected human chondrocytes(P0) expressing MMP2 short-GFP, transfection rate 15%, showingfunctionality of the cloned promoter-reporter construct. Image taken byfluorescence microscope with a 470/40 nm excitation filter, a 495 nmbeamsplitter and a 525/50 nm emission filter.

FIG. 24, Amaxa Nucleofector™ technology transfected human chondrocytes(P0) expressing MMP2 long-GFP, transfection rate 17%, showingfunctionality of the cloned promoter-reporter construct. Image taken byfluorescence microscope with a 470/40 nm excitation filter, a 495 nmbeamsplitter and a 525/50 nm emission filter.

MODES FOR CARRYING OUT THE INVENTION

Definitions

Bioinductive: refers to a natural or synthetic biomaterial thatinfluences cells in such a way to preserve or induce a differentiatedphenotype, even in the absence of exogenously added growth factors.Skelite™ is a good example for a bioinductive or osteoinductivematerial.

Non-destructive/non-invasive: refers to an assay and allows themeasurement of key parameters without destroying the current cellculture.

Real-time: refers to a direct measurement of signals produced byreporter molecules related to the cell-based assay described in thecurrent invention

3D: refers to a cell culture system where cells are kept in athree-dimensional condition to provide a tissue-like environment andtherefore allows to preserve or induce a differentiated phenotype of thecells.

3D micro-cultures: refers to three dimensional cell culture conditionsoptionally including biomaterials or scaffolds where cells are kept in atissue-like environment which preserves or induces a differentiatedphenotype of the cultivated cells and requires only a limited amount ofcells in order to qualify for high throughput applications.

2D: refers to the expansion of cell cultures in an anchorage dependentcondition on the surface of a plastic or any other biomaterialsubstrate.

Promoter-reporter constructs: are various constructs where a promoter ora transcriptional element thereof is linked to reporter molecules suchas green fluorescent protein or luciferase to perform real-time andnon-destructive measurements in cell cultures.

Skelite™ (Millenium Biologix Inc., Canada): is a synthetic bioactivebone biomaterial on basis of calcium phosphate. This exceptionalbiological performance is based on a chemical composition and physicalstructure that mimics natural bone.

Read-out: in the present context, the term read-out is used forqualitative and quantitative assessment of signals produced by reportermolecules that are e.g. detected by a conventional standard fluorescenceplate reader or a fluorescence microscope. Since cell culture parametershave been adapted to various multiwell plates, easy and convenientread-out through conventional standard plate readers has been achieved.This allows statistical determination of parameters such as accuracy,reproducibility and detection limit. These are important aspects for theadaptation to high throughput systems in drug screening applications.

GFP: in the present context, the term “green fluorescent protein” isintended to indicate a protein which, when expressed by a cell, emitsfluorescence upon exposure to light of the correct excitation wavelength(Chalfie et al. 1994).

Luminophore: the luminophore is the component that allows to bevisualized and/or recorded by emitting light related to the degree ofinfluence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel cell-based screening assay onbasis of gene-reporter technology and variants thereof that mayoptionally include biomaterials and scaffolds, either natural orsynthetic ones, to mimic the environmental nature of a given tissuewithin cell culture conditions on a small-scale level. Thus, morefunctional screening of cellular events will be feasible on a highthroughput level. Most preferably, the disclosed cell culture conditionsin combination with transfected cells are suitable to cultivate cellsderived from musculoskeletal tissues or cells being able todifferentiate in such tissues.

While growth and maintenance of cells in 3D culture provide cell-celland cell-matrix interactions as they occur in vivo, growth of cells in2D culture lacks these interactions and represents therefore a ratherartificial situation. Hence, such culture conditions are only suitablefor proliferation studies and not for experimental conclusions requiringa differentiated phenotype. Although this is well known, most commercialcellular assays including gene-reporter assays are based on cultivationof cells on plastic culture flasks. In limited cases it may becomepossible to improve the experimental situation of 2D cultures by simplyreplacing the artificial plastic culture substrate by a thin layer of amore appropriate biomaterial. This may then allow to circumvent the needof much more complex 3D cell culture conditions. A good example for thisis the Osteologic™ (Millenium Biologix Inc., Canada) bone cell culturesystem which consists of sub-micron synthetic calcium phosphate thinfilms coated onto various culture vessels. Thus, it has beendemonstrated to assess osteoclast/osteoblast activity and growth in amore biological relevant manner. Recent studies have shown that beyondbone cells also chondrocytes grow significantly better on Osteologic™substrate than on traditional tissue culture plastic.

Another important aspect of the present invention are various 3D cultureconditions allowing to screen cellular behavior of cells, mostpreferably those of musculoskeletal tissue or precursor cells underconditions that strongly support differentiation along a desired lineagepathway or maintain the corresponding fully differentiated phenotypeover an extended time in culture. By transfecting the mentioned celltypes with a corresponding gene-reporter construct such as described inthis invention, monitoring of cell differentiation and commitment to therespective cell lineage is possible. Such screening assays will also aidin the development of drug candidates or drug targets by elucidating thefunction of those drugs or genes during differentiation along thelineage pathway. Alternatively, by using already differentiated cells ofhealthy tissue and comparing those with cells from pathological tissuesuch as cells derived from tissues of arthritic joints, highly efficientscreening of drugs can be achieved. The 3D culture system of theembodiment may include some natural or synthetic scaffold material likealginate, agarose, hyaluronic acid, Skelite™ or any other materialproviding a three dimensional structure where cells can communicate witheach other via autocrine and paracrine factors as well as have theappropriate feedback from extracellular substances such as they occur invivo.

The invention also encompasses the downscale or adjustment of thedescribed 3D culture conditions to multiplate culture formats, e.g. 96or 384-multiwells, such as these 3D culture conditions for cellularscreening may contain only a few cells up to a couple of thousands perculture system and qualify for high throughput applications by providinga readily machine readable signaling with e.g. standard plate readers.This is of major advantage since 3D cultures often require a largeamount of cells and are therefore per se not applicable for efficientscreening of agents, if biological more relevant primary cell sourcesshall be the basis for cellular screening.

The current invention will therefore provide a platform for small-scale3D tissue cell culture systems by combining proper biomaterials togetherwith key marker promoter-reporter constructs. Through this downscalecostly culture material, cells and testing substances, e.g. growthfactors, hormones or any other culture media components, can be saved.Another major benefit is the possibility to adjust such small-scaleon-line and nondestructive screening assays to commonly used multiwellplates (96 to 384-well plates) to achieve fast, simple and convenient“reporter read-out” by means of conventional standard plate readersbesides other analytical tools, e.g. a fluorescence microscope.

The current invention also provides a new set of human promoter-reporterconstructs that can be used to transfect primary cells, cell lines oreven to prepare transgenic animals such as transgenic mouse lines thatexpress the reporter under the control of a promoter. The scope of thispart of the invention is described in the following by means of a fewexamples.

The current invention provides a method for screening agents ascandidates for drugs or growth factors for enhancing the formation ofnew cartilage tissue in vitro. Cells transfected with a constructcomprising the human COL2 or equivalent thereof, e.g. syntheticequivalent thereof, or in combination with the human COLL promoter orany other promoter element ligated to distinct reporter gene, arecultivated in 3D systems and treated with an activator of the COL2promoter. The agent being screened may be tested for its ability tostimulate the COL2 promoter. The agent is a candidate as a drug orsource of a drug being able to induce collagen type II expression invitro and to increase new tissue formation. While collagen type II isbeing stimulated by the screened drug, COL1 expression in contrast willbe reduced since it is a negative marker for the differentiatedphenotype of hyaline cartilage. While a recent paper published byBergwitz et al., 2001 describes the transfection of chondrocytes with arat promoter of collagen type II, one has to realize that promoterelements may be very species specific. While some elements of the COL2promoter will be necessary in the rat some of the same regulatoryelements will not be needed in human or vice versa. Therefore, even ifhighly identical sequential regions exist on both promoters they maybehave in a complete different way in vivo. This is fundamental and mayhave strong effects when agents are screened in cultures and may preventthe discovery of new lead compounds or negatively influence leadoptimization that may not be obvious. This invention thereforepreferably applies to the use of human promoter elements transfected inthe background of human cells.

In accordance with a further embodiment the invention provides a methodfor the assessment of the chondrocyte phenotype by using promoterelements of the human SOX9 and COMP genes. Both genes are chondrogenicmarkers that can be used to indicate chondrogenic differentiation ofprecursor cells or to detect the recovery and maintenance of thedifferentiated phenotype of articular chondrocytes follwowing expansionin 2D culture. Cell cultures transfected with the abovepromoter-reporter constructs will be screened with agents that mayinduce and/or maintain the differentiated phenotype in vitro.

The current invention discloses also a method for screening agents ascandidates for drugs for prophylaxis or treatment of mammalian disorderscaused or mediated by aggrecanase-1 (ADAMTS4) expression. Thus, cellsmay be transfected in a cell background that strongly inducesaggrecanase-1 expression e.g. rheumatoid or osteoarthritic cell sourcestransfected with a construct containing a transcriptional promoterelement from the human aggrecanase-1 gene or equivalent thereof, e.g.synthetic equivalent thereof, ligated to a reporter gene and cultivatedas 3D micro cultures optionally on/within a biomaterial/scaffold.Another experimental set-up would include healthy chondrocytic cellstransfected with a construct containing a transcriptional promoterelement from the human aggrecanase-1 gene or equivalent thereof, e.g.synthetic equivalent thereof, ligated to a reporter gene and cultivatedas 3D microstructures optionally on/within a biomaterial/scaffold andtreated with an inducer of the aggrecanase-1 promoter activity e.g.interleukin 1. The agent being screened is then tested for its abilityto suppress promoter activity. The agent is a candidate as a drug orsource of a drug for prophylaxis or treatment of mammalian disorderscaused or mediated by aggrecanase-1 expression if the agent reducesstimulated promoter activity. Where an agent is determined to inhibitstimulation of aggrecanase-1 promoter, this indicates a higherlikelihood of inhibiting any degradation of cartilage matrix.

The current invention discloses also a method for screening agents asdrug candidates for prophylaxis or treatment of mammalian disorderscaused or mediated by expression of matrix metalloproteinases (MMPs).MMP's e.g. MMP2 play an important role in the evolution of jointerosions in patients with non-inflammatory osteoarthritis andinflammatory rheumatoid arthritis. The gelatinase MMP2 has further shownto be involved in cancer, above all in tissue-invasive metastaticdiseases.

MMP promoter elements linked to reporter molecules like e.g. GFP canthus be used not only to study cartilage degenerative processes takingplace in arthritic conditions but also can be applied to study theobstacles of cancer development and progression via metastasisformation. In both processes the degradation of the extracellular matrixis taking place and this process can be best studied by using biologicalrelevant cell culture conditions where the cells behave similar to thein vivo situation. Cells may then be transfected into a cell backgroundthat strongly induces MMP expression e.g. rheumatoid or osteoarthritic.or tumor cell sources, with a construct containing a transcriptionalpromoter element from the human MMP2 gene or equivalent thereof, e.g.synthetic equivalent thereof, ligated to a reporter gene and cultivatedas a 3D micro culture optionally on/with a biomaterial/scaffold. Thiscell culture system may then be screened with agents that may suppressthe expression of the reporter molecule, indicative for a molecule thatwill allow to reduce MMP expression also in vivo. Another experimentalset-up would include e.g. primary human cells isolated from healthycartilage tissue and transfected with a construct containing atranscriptional promoter element from the human MMP gene or equivalentthereof, e.g. synthetic equivalent thereof, ligated to a reporter geneand grown optionally on/within a biomaterial/scaffold and treatedrespectively with an inducer of MMP promoter activity e.g. TumorNecrosis Factor α. The agent being screened is then tested for itsability to suppress stimulation of the promoter and a potentialcandidate as a drug or source of a drug for prophylaxis or treatment ofmammalian disorders from cartilage degeneration.

While the reporter molecule will preferentially be firefly luciferaseand GFP or any other fluorescence molecule, other reporter systems foruse for this purpose include, for example beta-galactosidase gene(beta.gal) and chloramphenicol and acetyltransferase gene (CAT). Assaysfor expression produced in conjunction with each of these reporter geneelements are well-known to those skilled in the art. By preferentiallyhaving luciferase and GFP as reporter molecule, the advantage is ofbeing able to perform real-time follow-up studies on cell cultureswithout the need to destroy the cells. A further advantage by havingreporter molecules that allow nondestructive measurement is to be ableto perform temporal and spatial analysis, a topic that is of majorrelevance when tissue-engineered constructs are grown in vitro. Thisallows monitoring cell relevant marker gene expression in these cellculture systems in a real-time and non-destructive manner and todetermine whether the cells in the grown tissue are equallydifferentiated and well nourished. Especially when having 3D culturesthat are cultivated over an extended time, e.g. four weeks as it is thecase in the field of cartilage tissue-engineering, it may be of greatadvantage to follow the development of the same tissue in vitro withoutdestroying the material.

The current invention does not only cover the aspect of having singlepromoter-reporter elements in one cell. The combination of severalvectors containing one or more promoter elements in the same cells e.g.cotransfection with cDNA libraries in the same cell may also bedisclosed. This may be of major importance when screening new proteinsthat may act as inductors or repressors on the reporter construct to betested. In such a screening process libraries containing expressionvectors where cDNA are linked to a constitutive promoter like e.g. CMVmay be co-transfected with to be analyzed promoter-reporter constructand screened for the induction or repression of the reporter molecules.This will allow to detect new target molecules e.g. transcriptionfactors and to identify new lead compounds for clinical applications.

The current invention also encompasses cell lines that are derived fromthe above mentioned transfection or co-transfection experiments, thesecell lines can then be used as standard elements during furtherscreening processes for the discovery of new molecules.

The current invention has disclosed a novel cell-based screening toolthat may be applicable for screening of drugs, growth factors or anyother beneficial components during development of cellular ortissue-engineered therapies. It does not matter whether the donor cellsare from an autologous, allogeneic, xenogeneic cell source or whetherthe cells are non-differentiated precursor cells or already fullydifferentiated cells. Furthermore, an in vitro screening system thatallows to be performed on miniaturized 3D tissue-like cultures has notbeen disclosed before and enables a more reliable validation of cellulartargets, to assess more precisely toxicological responses and toincrease the probability of success of new leads in the clinic.

While U.S. Pat. No. 6,200,760, U.S. Pat. No. 6,083,690 and U.S. Pat. No.6,338,944 describe methods of screening agents in combination with genepromoter-reporter constructs, these inventions have not properlyaddressed the biological relevance of any cellular screening event,above all in context with the important issue to simultaneously allowaccurate and reliable read-out on a higher throughput level. Otherpatents like U.S. Pat. No. 5,858,721 have disclosed the method of 3Dsystem cultivation for tissue-engineering applications but have notconsidered the application of using such systems within micro scalecultures to be used within 96 or 384-well

Therefore, applications may even include the possibility to screen thetoxicity of new chemicals and drugs as an important alternative toanimal models for e.g. the cosmetic industry. By cultivating cellpopulations in a three dimensional system new drugs or molecules can betested more thoroughly since a tissue-like system is provided.Cell-based screening tools may be the preferred technique in drugdiscovery, because it generates leads with a higher probability ofprogressing to clinical trials. Another important aspect includes thedetermination of dose response curves for new drugs and can be useful inthe field of pharmakokinetics. Cells isolated from a patient andcultured under 3D conditions disclosed in the invention may then be usedto assess further treatment by choosing the best of a selection of drugmolecules. Furthermore, cells can be isolated on later stage and checkedfor disease progression. Therefore, the current invention relates to theapplication for cell-based diagnostics.

Another important aspect of the current invention is the use of thescreening assay as a quality control for cell/tissue-based therapies forproduct and material testing. Because compendial methods do not yetexist, meaningful assays are required and need to be validated tomonitor performance of key components such as the cell source or anybiomaterial to be included. The herein described assay may be especiallysuitable for determining the cell potency of any cell source, e.g.autologous, allogeneic, xenogeneic or genetically engineered cells. Acritical test could be to ascertain the necessary proliferative and/ordifferentiation capability of the cell source. Within any celltherapeutic approach such as autologous chondrocyte implantation (ACI)it would be possible to monitor the differentiation ability after lotrelease of the product and to better control or assess the clinicaloutcome of such a therapy. In case of a tissue-engineered product thatrequires further cultivation in 3D for a certain time period followingcellular expansion, a screening assay of this invention could be used,e.g. along with other quality control tests as a checkpoint for lotrelease of the final implant.

Yet another important aspect of the described invention is the use ofthis screening assay as a diagnostic tool. In this sense, donor cellsfrom autologous, allogeneic, or xenogeneic sources, e.g. healthy livingadults, fetals, and/or cadavers may be checked for their suitability(cell potency) within a cell/tissue-engineered therapy. Further, thecorresponding cells may be analyzed in the clinic for theirproliferative and/or differentiation ability in order to decide on themost promising therapy. This may be a cellular therapy, atissue-engineered therapy, or in case of a negative diagnosis with thedisclosed assay a traditional surgical approach. It may also be the casethat the diagnostic assay will monitor the cellular status of the donorsource and in case of any pre-determined deficiencies correct these bye.g. adding the required growth factor(s), hormones, pharmaceuticalagent etc.

A further application of the current invention may include theassessment of the performance of biomaterials in combination with cellsor tissue. Cells or tissues containing transfected cells withappropriate promoter-reporter constructs may be used to assess theinductive potential of biomaterials regarding their potential ofinducing new tissue formation or preserving the differentiatedphenotype. Biomaterials that will positively influence the cultivatedcells with respect to inducing differentiation or preserving the correctphenotype may show a higher expression of the reporter moleculeaccording to the selected marker promoter. This may then be indicativeof a positive feedback of the material to the cell and will help tobetter design and adapt new materials to the corresponding cells ortissue.

In yet another aspect, the biomaterials coated with a key markerpromoter-reporter construct may be used to assess the degradation orresorption of the biomaterial in vivo or in vitro. When biomaterials areresorbed in vivo or in vitro plasmid released from the material willtransfect surrounding cells. If an adequate promoter-reporter moleculeis used the surrounding cells will then express a reporter molecule e.g.GFP indicative for the released vector molecules and resorption anddegradation can be studied.

The current invention may also be used to study new in vitro tissueformation on a larger scale by using transfected cells with selectedpromoter-reporter molecules. These cells may then be grown in vitro orin vivo and tissue formation can be assessed by determining theexpression of the reporter molecule. A similar experimental setup may beused and performed in animal model, were transfected cells withcorresponding promoter-reporter constructs may be included in thetransplanted tissue to follow the development of the tissue in vivo.

The invention is now further described by means of examples.

EXAMPLE 1

3D Micro Cell Culture Models Mimicking a Cartilage Tissue-LikeEnvironment

Useful for 3D culture conditions that can be downscaled to e.g. 96 or384-well format, suitable for e.g. high throughput screeningapplications or to be applied as a quality control tool withincell-based therapies.

Cell Isolation and Propagation

Articular cartilage was harvested from healthy young (6 months) pigs orhuman donors (age 56 and 79 years). Minced cartilage pieces weredigested with 0.025% (weight/volume) collagenase and 0.015%(weight/volume) pronase in DMEM/F-12 containing 5% fetal calf serum(FCS), 73 μg/ml ascorbic acid, 100 IU/100 μg/ml penicillin/streptomycin,1 μg/ml insulin, 50 μg/ml gentamycin, 1.5 μg/ml amphotericin B, 2.5%Hepes buffer for 16 hours at 37° C. in 5% CO₂. Isolated chondrocyteswere spun, resuspended in complete medium, counted and plated at adensity of 5×10⁶ cells per cm². Cells were routinely passaged atconfluence (every 5-7 days). Proliferation medium was DMEM/F-12containing 10% FCS, 14.5 μg/ml ascorbic acid and 50 IU/50 μg/mlpenicillin/streptomycin.

Transfection and 3D Cell Culture Conditions

To prepare transfected cells that will be used for micro 3D cultures,three different transfection methods were applied.

a) Viral Infection Using Adenovirus (AV)

5×10⁴ chondrocytes per 24-well were infected with AV (MOI=100)containing GFP under the control of a CMV promoter for 16 hours inDMEM/F-12 containing 2% FCS, 14.5 μg/ml ascorbic acid and 50 IU/50 μg/mlpenicillin/streptomycin.

b) Amaxa Nucleofector™ technology

Human chondrocytes were transfected with pGFP-CMV using AmaxaNucleofector™ technology. Briefly, 5 μg plasmid were mixed with 5×10⁵cells in 100 μl nucleofection solution and subsequently nucleofectedusing program U-24 from Amaxa Nucleofector™ technology. Transfectedcells were plated in a 6-well plate, medium was changed after 24 hours.

-   -   c) Lipid Based Transfection Method Using Fugene6

2.5×10⁵ cells were transfected in a 6-well plate using Fugene6, Roche,Switzerland, with a plasmid containing the luciferase gene under thecontrol of a collagen type I promoter, kindly provided by F. Ramirez,New York. 3 μl reaction reagent per 1 μg DNA was used. Transfectionreagent was removed after 24 hours. To determine transfection rate, aco-transfection with pGFP-CMV was performed.

Subsequently, cells were detached from monolayer culture and put in oneof the following tissue-like culture cultivation methods describedbelow. In all cases, cells were then maintained in differentiationmedium DMEM/F-12 with 10% FCS, 1 μg/ml Insulin, 73 μg/ml ascorbic acidat 37° C. and 5% CO₂. Untransfected chondrocytes were used as control.

Monitoring GFP Expression in 3D Cultures

Transfected cells, e.g. porcine chondrocytes were qualitativelymonitored using a Zeiss Axiovert 25. The cells were illuminated with a50W HBO arc lamp. In the light path was a 470/40 nm excitation filter, a495 nm beamsplitter and a 525/50 nm emission filter. Images were takenusing Kodak EDAS290 directly mounted to the microscope. For quantitativemeasurement of expression intensity, transfected chondrocytes weremeasured with BMG Fluostar optima plate reader using 485/20 nmexcitation filter and 535/20 emission filter.

Monitoring Luciferase Expression in 3D Cultures

Transfected cells, e.g. porcine chondrocytes were monitored using aBerthold Detection System MPL2 luminometer. Expression intensity ofluciferase was measured 5 minutes after adding 100 μl PBS and 100 μlPromega's Bright-Glo™ reagent per 96-well for 10. seconds.

Monitoring Calcein-AM/Propidium Iodide Stained Cells

Cells were stained using 1 μg/ml calcein-AM and 1 μg/ml propidium iodidein phosphate buffered saline (PBS) per 2×10⁴ cells for 10 minutes. Thecells were illuminated with a 50 W HBO arc lamp. In the light path was a470/40 nm excitation filter, a 495 nm beamsplitter and a 525/50 nmemission filter to monitor life cells (green) and a 535/50 nm excitationfilter, a 580 nm beamsplitter and a 590LP nm emission filter to monitordead cells (red). Images were taken using Kodak EDAS290 directly mountedto the microscope.

Micro 3D Tissue-Like Culture Method 1-Alginate Discs

1×10⁵, 1.5×10⁵ or 2×10⁵ AV infected porcine chondrocytes from passage 2(P2), with a transfection rate of 90%, containing the GFP gene under thecontrol of a CMV promoter were spun and suspended in 80 μl 1.2% alginateKeltone LV dissolved in 0.9% NaCl, seeded into 96-well plates pre-coatedwith a 0.1 M CaCl₂-soaked isopore polycarbonate membrane filter(Millipore, Switzerland) and let be polymerized for 75 minutes at roomtemperature. Alginate discs were cultivated in differentiation medium asdescribed above. GFP expression intensity was measured during 12 daysusing BMG Fluostar optima. Significant GFP expression can be measuredduring 6 days for all cell numbers used, example with 2×10⁵ cells perwell can be seen in FIG. 1. GFP expression correlates with increasingcell number as can be seen e.g. on day 2 of the experiment, FIG. 2.Simultaneously, alginate discs were monitored visually with ZeissAxiovert 25, FIG. 3. At the last day of the experiment, day 12, cellswere tested for viability using calcein AM and propidium iodidestaining. Over 90% viability could be observed, FIG. 4.

Micro 3D Tissue-Like Culture Method 2-Agarose Discs

a) 1×10⁵, 1.5×10⁵ or 2×10⁵ AV infected P2 porcine chondrocytes,containing the GFP gene under the control of a CMV promoter weresuspended in 20 μl DMEM/F-12, mixed with 2% agarose (low-melting, Fluka)kept at 45° C. and pipeted quickly into 96-well plates and let be gelledfor 10 minutes at 4° C. Agarose discs were cultivated in differentiationmedium as described above. GFP expression intensity was measured during13 days using BMG Fluostar optima. Significant GFP expression can bemeasured during at least 7 days for all cell numbers used, example with2×10⁵ cells per well can be seen in FIG. 5. GFP expression correlateswith increasing cell number as can be seen e.g. on day 2 of theexperiment, FIG. 6. Simultaneously, agarose discs were monitoredvisually with Zeiss Axiovert 25, FIG. 7. At the last day of theexperiment, day 13, cells were tested for viability using calcein AM andpropidium iodide staining. Over 90% viability could be observed, FIG. 8

b) 1×10⁵, 1.5×10⁵ or 2×10⁵ with Amaxa Nucleofector™ technologytransfected P2 human chondrocytes with a transfection rate of 40%containing the GFP gene under the control of a CMV promoter were seededin agarose and measured for GFP expression intensity during 10 days asdescribed above. Significant GFP expression can be measured during 8days for all cell numbers used, example with 2×10⁵ cells per well can beseen in FIG. 9. GFP expression correlates with increasing cell number ascan be seen e.g. on day 2 of the experiment, FIG. 10. At the last day ofthe experiment, day 10, cells were tested for viability as describedabove. Over 90% viability could be observed.

-   -   c) Alternatively, 1×10⁴, 3×10⁴, 5×10⁴ or 7×10⁴ with Fugene6        transfected P2 porcine chondrocytes containing the luciferase        gene under the control of a COL1 promoter were seeded in agarose        as described above. Luciferase expression intensity was measured        as described in ‘Monitoring luciferase expression in 3D        cultures’ at day 1. Transfection rate was 15%, determined as        described above. FIG. 11 shows that luciferase expression        correlates with increasing cell number and that only 1500        transfected cells are needed to obtain statistically relevant        data. Cells were tested for viability using calcein AM and        propidium iodide staining. Over 90% viability could be observed.

Micro 3D Tissue-Like Culture Method 3-polyHEMA

96-well plates were coated 24 hours before use with 64 μl/cm² 10%polyHEMA (Polysciences, Europe GmbH) in 95% ethanol and let be dried insterile environment over night. 1×10⁵, 1.5×10⁵ or 2×10⁵ transfected P2porcine chondrocytes containing the GFP gene under the control of a CMVpromoter were seeded into pre-coated 96-well plates and cultivated andmeasured for GFP expression intensity during 10 days as described above.Significant GFP expression can be measured during 6 days for all cellnumbers used, example with 2×10⁵ cells per well can be seen in FIG. 12.GFP expression correlates with increasing cell number as can be seene.g. on day 3 of the experiment, FIG. 13. Simultaneously, cells onpolyHEMA were monitored visually with Zeiss Axiovert 25, FIG. 14. At thelast day of the experiment, day 10, cells were tested for viability asdescribed above. Over 90% viability could be observed.

EXAMPLE 2

Useful for the automated production of 3D micro cell cultures that canbe used to study promoter-reporter events in biological relevanttissue-like environment using high throughput screening applications.

A suitable cell line, e.g. primary chondrocytes is expanded until thenumber of required cells is obtained. Cells are transfected using one ofthe methods described in example 1 with the promoter-reporter constructof interest, e.g. GFP under the control of COL2. Transfected cells aredetached and put in a downscaled version of any of the 3D tissue-likeculture systems described in example 1 using a pipeting robot. The cellsolution is e.g. mixed in a ratio 1:1 with 2% agarose at a temperatureof 45° C. and pipeted into a 384-well plate. For polymerization theplate is incubated for 10 minutes at 4° C. Subsequently, the plate iscultivated under standard differentiating culture conditions asdescribed in example 1. Factors or components of the extracellularmatrix, which promote the process of growing and differentiating, areadded and exposed to e.g. a differentiating medium. Plates are measuredautomatically for GFP expression intensity using a standard fluorescenceplate reader at time points of interest. Expression profile givesinformation about which factors or components enhance or repressextracellular matrix formation, respectively.

Any of the 3D tissue-like cell culture methods described in example 1 issuitable for downscaling and to be used within automated high throughputscreening applications. Alginate solution containing transfected cellsmay be pipeted in 384-well plates containing isopore polycarbonatemembrane membranes (Millipore, Switzerland) soaked with 0.M CaCl₂ at thebottom. To seed transfected cells on polyHEMA (Polysciences, EuropeGmbH) pre-coated 384-well plates may be used.

For all systems, untransfected cells are used as control.

EXAMPLE 3

Clinical Quality Control Tool for the Assessment of Cell-Based Therapies

Useful e.g. as quality control and diagnostic tool for cell culturesused within cell-based therapies, like e.g. autologous chondrocytetransplantation (ACT) or quality assurance of in vitro engineeredconstructs.

a) Human cells derived from a patient's tissue, e.g. cartilage areexpanded and treated according to the cell-based therapy used. Analiquot of said cells is taken to gain knowledge about e.g. chondrogenicpotential, i.e. re-differentiation of chondrocytes or the necessity ofadditional treatment. Cells of the taken aliquot are then transfectedwith one of the methods described in example 1 with a key markerpromoter-reporter construct, e.g. COL2-GFP to monitor redifferentiationin chondrocytes, and are cultivated in the appropriate 3D microtissue-like culture system. From grown constructs chondrogenic potentialis assessed measuring GFP expression intensity using a standardfluorescence plate reader. The result, combined with e.g. cell viabilityreveals information about the chondrogenic potential and/or whetheradditional treatment e.g. factor adding or a complementing therapy isrequired.

b) To assess quality and characteristics of the cells used during invitro production of tissue-engineered e.g. cartilage like constructs analiquot of the proliferated cells is transfected with one of the methodsin example 1 with a key marker promoter-reporter construct e.g.COL2-GFP. Subsequently the cells are cultured separately but in parallelin an appropriate 3D micro tissue-like culture system and GFP expressionintensity is monitored. The chondrogenic potential is assessedaccordingly and correlated with previously defined process-relevantconditions. The correlation gives information whether the to be producedconstructs fulfils the required specifications.

EXAMPLE 4

cDNA Expression Library Screening Platform Using 3D Micro Tissue-LikeCell Cultures

Useful for screening of cDNA expression libraries in 3D microtissue-like cell culture environment.

In order to find e.g. an inducer of the collagen type II gene,CMV-driven cDNA expression libraries of interest are co-transfected witha plasmid containing the promoter of collagen type II in front of theluciferase gene into a selected cell line. The cells are cultivated inone of the 3D micro tissue-like cell culture models as described inexample 1 in e.g. 96-well plates and subsequently screened forluciferase expression intensity. DNA plasmid isolation from cells thatshow highest luciferase expression is performed. Obtained DNA istransformed into bacteria and amplified. Plasmid is isolated andco-transfected again, the screening for highest luciferase expression isrepeated. This cycle may be performed several times to be sure toisolate only plasmid containing cDNA of interest. cDNA on purifiedplasmid is sequenced and gene that influences promoter of interest maybe identified.

EXAMPLE 5

Useful for monitoring influence of various drugs on primaryosteoarthritic cell samples, e.g. osteoarthritic chondrocytes.

Cells, e.g. osteoarthritic human chondrocytes are infected with a keymarker promoter-reporter construct for osteoarthritis, e.g.aggrecanase-1 (ADAMTS4)-GFP or MMP2-GFP using a viral system tocircumvent known difficulties with plasmid transfection, FIG. 15 showshighly transfected osteoarthritic chondrocytes (P2) using AV withCMV-GFP. Infected cells are seeded into a 96-well plate treated withhypothetical factors and components to assess their potential inosteoarthritis treatment, i.e. down-regulation of e.g. aggrecanase-1 orMMP2 expression. Plates are measured for GFP expression intensity usinga standard fluorescence plate reader. Effectiveness of factors andcomponents tested can be correlated to GFP signal intensity, i.e. lowGFP signal equals highly efficient osteoarthritic treatment.

EXAMPLE 6

Chondrocyte Cell Culture on Osteologic™

Human chondrocytes isolated from sequential enzymatic digestion of aknee biopsy were cultured in DMEM/F12 supplemented with 10% FCS and 100IU/100 μg/ml penicillin/streptomycin. Cells were passaged once in T80Falcon flasks harvested and seeded onto Osteologic™ discs in 24 wellplates at 1×10⁴ cells per well at passage 2 (P2). Control wells wereseeded directly into plastic wells without Osteologic™ discs on the sameplate. Parallel plates were prepared for a time course study with cellcounts taken at 0, 2, 4 & 7 days. Cells were trypsinized and counted byhemocytometer using the trypan blue method, FIG. 16. A second set ofplates was plated with the same cells after culturing in flasks for anadditional passage (P3). Plates were cultured for 7 days fixed with 1%gluteraldehyde in PBS and stained with a combined Periodic Acid Schiffstain (PAS) and alcian blue stain for detection of proteoglycans, FIG.17.

EXAMPLE 7

Human Promoter for Detection of Sox9 Expression

Useful for monitoring of sex determining region (SRY)-box containinggene 9 (SOX9) expression. SOX9 is expressed during redifferentiation inchondrocytes in 3D tissue-like culture systems.

A 750 bp fragment of the SOX9 promoter (GenBank accession number:AB022194) as described by Kanai and Koopman, 1999 is amplified withprimers SOX9 sense (SEQ ID NO 1) and SOX9 antisense (SEQ ID NO 2) forgenomic PCR according to standard protocols. The PCR product is digestedwith restriction enzymes HindIII and KpnI and ligated into pEGFP-1(Clontech, Switzerland, GenBank accession number U55761) or intopGreenLantern (Gibco, Switzerland) digested with HindIII and KpnI. Thisproduces a plasmid containing GFP under the control of a SOX9 promoter.

The resulting plasmid is transfected into a suitable cell line, e.g.passage 0 (P0) human chondrocytes and SOX9 expression is monitored. FIG.18 shows that the constructed plasmid is functional.

EXAMPLE 8

Human Promoter for Detection of Collagen Type Expression

Useful for monitoring of collagen type I (COL1) expression. COLL isexpressed during dedifferentiation in chondrocytes in 2D culturesystems.

A 450 bp fragment of the α2(I) collagen promoter (COL1) (GenBankaccession number: AF004877) as described by Inagaki et al., 1994 isamplified from a plasmid kindly provided by F. Ramirez, New York withprimers COLL sense (SEQ ID NO 3) and COL1 antisense (SEQ ID NO 4)according to standard PCR protocols. The PCR product is digested withrestriction enzymes BglII and EcoRI and ligated into pEGFP-1 (Clontech,Switzerland, GenBank accession number U55761) or into pGreenLantern(Gibco, Switzerland) digested with BglII and EcoRI. This produces aplasmid containing GFP under the control of a COLL promoter.

The resulting plasmid is transfected into a suitable cell line, e.g. P0human chondrocytes and COL1 expression is monitored. FIG. 19 shows thatthe constructed plasmid is functional.

EXAMPLE 9

Human Promoter for Detection of Collagen Type II Expression

Useful for monitoring of collagen type II (COL2) expression. COL2 isexpressed during redifferentiation in chondrocytes in 3D tissue-likeculture systems.

A 3.785 kb fragment of the α2(I) collagen promoter (COL2) as describedby Ghayor et al., 2000 is cut out from a plasmid kindly provided by L.Ala-Kokko, Oulu, Finland with restriction enzyme PdiI. The obtainedfragment was ligated into pEGFP-1 (Clontech, Switzerland, GenBankaccession number U55761) or into pGreenLantern (Gibco, Switzerland)digested with PdiI. This produces a plasmid containing GFP under thecontrol of a COL2 promoter.

The resulting plasmid is transfected into a suitable cell line, e.g. P0human chondrocytes and COL2 expression is monitored. FIG. 20 shows thatthe constructed plasmid is functional.

EXAMPLE 10

Human Promoter for Detection of Cartilage Oligomeric Matrix Protein(COMP) Expression

Useful for monitoring of cartilage oligomeric matrix protein (COMP)expression. COMP is expressed during redifferentiation in chondrocytesin 3D tissue-like culture systems.

A 750 bp fragment of the COMP promoter (Genank accession number:AF069520) as described by Deere et al., 2001 is amplified with primersCOMP sense (SEQ ID NO 5) and COMP antisense (SEQ ID NO 6) for genomicPCR according to standard protocols. The PCR product is digested withrestriction enzymes HindIII and BamHI and ligated into pEGFP-1(Clontech, Switzerland, GenBank accession number U55761) or intopGreenLantern (Gibco, Switzerland) digested with HindIII and BamHI. Thisproduces a plasmid containing GFP under the control of a COMP promoter.

The resulting plasmid is transfected into a suitable cell line, e.g. P0human chondrocytes and COMP expression is monitored. FIG. 21 shows thatthe constructed plasmid is functional.

EXAMPLE 11

Human Promoter for Detection of Aggrecanase-1 Expression

Useful for monitoring of aggrecanase-1 (ADAMTS4) expression.Aggrecanase-1 is expressed during degradation of cartilage extracellularmatrix, e.g. osteoarthritic chondrocytes.

A 1.2 kb fragment of the aggrecanase-1 promoter (GenBank accessionnumber: AB039835) as described by Mizui et al., 2000 is amplified withprimers aggrecanase sense (SEQ ID NO 7) and aggrecanase antisense (SEQID NO 8) for genomic PCR according to standard protocols. The PCRproduct is cloned into PCR-Blunt II-TOPO vector (Invitrogen,Switzerland). The newly generated plasmid is digested with restrictionenzymes HindIII and KpnI and the obtained aggrecanase-1 fragment isligated into pEGFP-1 (Clontech, Switzerland, GenBank accession numberU55761) or into pGreenLantern (Gibco, Switzerland) digested with HindIIIand KpnI. This produces a plasmid structed plasmid containing the 1.1 kbfragment is functional. FIG. 24 shows that the constructed plasmidcontaining the 1.7 kb fragment is functional.

While there are shown and described presently preferred embodiments ofthe invention, it is to be distinctly understood that the invention isnot limited thereto but may be otherwise variously embodied andpracticed within the scope of the following claims.

1. A screening method for compounds having a modulating effect oncellular development and/or cell differentiation and/or cellularprocesses, said method comprising the following steps: a) cultivatingcells harboring a promoter-reporter construct in a 3D micro-cultureunder conditions mimicking the natural in vivo environment (3Dtissue-like conditions) of said cells, or cultivating said cell in a 2Dculture on bioinductive material, b) contacting said cells with a testcompound and comparing the read-out of the promoter-reporter constructto a control.
 2. The method of claim 1, wherein said 3D tissue-likeconditions comprise either 3D aggregated cells, cultivated under highcellular density only, and/or cells cultivated with natural or syntheticscaffold/biomaterial.
 3. The method of claim 2, wherein saidscaffolds/biomaterials are a biomaterial substrate or scaffold thatpromotes normal physiological activity, in particularscaffolds/biomaterials selected from the group of naturalscaffolds/biomaterials consisting of alginate, agarose, hyaluronic acid,collagen, proteoglycan and mixtures thereof, or from the group ofsynthetic scaffolds/biomaterials consisting of Skelite™, polyHEMA,polyglycolic acid (PGA), polylactic acid (PLA) and mixtures of PGA andPLA.
 4. The screening method of claim 1, wherein said cells are derivedfrom healthy or pathological musculoskeletal tissues or precursor cellsbeing able to differentiate and form de novo musculoskeletal tissue,preferably said cells stem from humans.
 5. The method of claim 4,wherein said tissue is selected from the group consisting ofchondrocytes, bone cells, rheumatoid cells, osteoarthritic chondrocytes,stem cells, mesenchymal cells, cartilage or bone tumor cells.
 6. Thescreening method of claim 1, wherein said promoter is selected from thegroup consisting of human COL1, COL2, SOX9, COMP, MMP2, andaggrecanase-1 (ADAMTS4).
 7. The screening method of claim 1, whereinsaid reporter is selected from the group of GFP, luciferase,β-galactosidase, chloramphenicol acetyltransferase gene (CAT).
 8. Thescreening method of claim 1, wherein said cells stem from humans andsaid promoter-reporter construct comprises a reporter gene under controlof a human promoter wherein said promoter is selected from the groupconsisting of human COL1, human COL2, human SOX9, human COMP, humanMMP2, and human aggrecanase-1 (ADAMTS4) and said reporter gene encodes aprotein with an activity that can be detected by colorimetric orfluorescent methods, in particular said reporter is selected from thegroup consisting of GFP, luciferase, β-galactosidase, chloramphenicolacetyltransferase gene (CAT).
 9. The screening method of claim 1,wherein said cells comprise more than one promoter-reporter construct.10. The screening method of claim 1, wherein said test compounds areselected from the group consisting of chemical libraries, naturalproduct libraries, peptide libraries, cDNA libraries and combinatoriallibraries.
 11. The screening method of claim 1, wherein said method isperformed in a multiplate culture format e.g. 96 or 384-mulitwells. 12.The screening method of claim 11, wherein the 3D micro cultures areproduced in an automated fashion e.g. by robotic system.
 13. Thescreening method of claim 1, wherein said cells are contacted with anactivator or suppressor of said promoter and with a test compound. 14.The screening method of claim 1, wherein said method is used as aquality control tool to assess the chondrogenic potential of isolatedcells prior to implantation within cell-based therapies.
 15. Thescreening method of claim 1, wherein said method is used as a qualitycontrol tool to assess a process producing in vitro tissue-engineeredcartilage constructs usable for treatment of cartilage defects.
 16. Thescreening method of claim 1, wherein said method is used as a tool toassess the cell potency and such the suitability of cells for celltherapy and/or tissue engineered therapy.
 17. A method for producing atransgenic animal comprising transforming an animal with apromoter-reporter construct wherein said reporter is selected from thegroup consisting of GFP, luciferase, β-galactosidase, chloramphenicolacetyltransferase gene (CAT) and said promoter is selected from thegroup consisting of COL1, COL2, SOX9, COMP, MMP2, and aggrecanase-1(ADAMTS4).
 18. A transgenic animal comprising a promoter-reporterconstruct, wherein said construct comprises a reporter selected from thegroup consisting of GFP, luciferase, β-galactosidase, chloramphenicolacetyltransferase gene (CAT) and a promoter selected from the groupconsisting of COL1, COL2, SOX9, COMP, MMP2 and aggrecanase-1 (ADAMTS4).19. A cell line derived from the transgenic animal of claim
 18. 20. Amethod comprising using the transgenic animal of claim 18 in a screeningmethod for screening compounds having a modulating effect on cellulardevelopment and/or cell differentiation and/or cellular processes.
 21. ADNA construct for cell transfection comprising a reporter gene undercontrol of a human promoter wherein said promoter is selected from thegroup consisting of human COL1, human COL2, human SOX9, human COMP,human MMP2, and human aggrecanase-1 (ADAMTS4) and said reporter geneencodes a protein with an activity that can be detected by colorimetricor fluorescent methods.
 22. The DNA construct of claim 21, wherein saidreporter is selected from the group consisting of GFP, luciferase,β-galactosidase, chloramphenicol acetyltransferase gene (CAT).
 23. Acell comprising a reporter construct of claim
 21. 24. A cell linecomprising a reporter construct of claim
 21. 25. The cell or cell lineof claim 23, wherein said cells are derived from healthy or pathologicalmusculoskeletal tissues or precursor cells being able to differentiateand form de novo musculoskeletal tissue, preferably said cells stem fromhumans.
 26. The cell or cell line of claim 25, wherein said cells areselected from the group consisting of chondrocytes, bone cells,rheumatoid cells, osteoarthritic chondrocytes, stem cells, mesenchymalcells, cartilage or bone tumor cells.
 27. A method comprising performinga cellular screening assay with a cell line of claim
 24. 28. A methodcomprising using a cell of claim 23, for the in vitro formation oftissue.
 29. A method for testing whether a material has bioinductivecharacteristics, said method comprising the following steps: culturingcells harboring a promoter-reporter construct on the material to betested and comparing the read-out of the promoter-reporter construct toa control.
 30. The method of claim 29, wherein said cells are humancells.
 31. The method of claim 29, wherein said reporter is selectedfrom the group consisting of GFP, luciferase, β-galactosidase,chloramphenicol acetyltransferase gene (CAT) and the promoter isselected from the group consisting of human COL1, COL2. SOX9, COMP,MMP2, and aggrecanase-1 (ADAMTS4).
 32. A method for testing whether abiomaterial is degraded or resorbed in vivo or in vitro, said methodcomprising the following steps: culturing cells harboring apromoter-reporter construct on the material to be tested and monitoringexpression of the reporter gene in said cells.
 33. The method of claim32, wherein said cells are human cells.
 34. The method of claim 32,wherein said reporter is selected from the group consisting of GFP,luciferase, β-galactosidase, chloramphenicol acetyltransferase gene(CAT) and the promoter is selected from the group consisting of humanCOL1, COL2, SOX9, COMP, MMP2, and aggrecanase-1 (ADAMTS4).
 35. A methodfor the quality control of cells cultivated in vitro comprising:transfecting cells that have been cultured in vitro with a key markerpromoter-reporter construct and cultivating said transfected cells in a3D culture and detection of the reporter read-out which is indicativefor differentiated cells.
 36. The method of claim 35, wherein said cellsare selected from the group consisting of chondrocytes, bone cells,rheumatoid cells, osteoarthritic chondrocytes, stem cells, mesenchymalcells, cartilage or bone tumor cells, the promoter is selected from thegroup consisting of GFP, luciferase, β-galactosidase, chloramphenicolacetyltransferase gene (CAT) and the promoter is selected from the groupconsisting of human COL1, COL2, SOX9, COMP, MMP2, and aggrecanase-1(ADAMTS4).